Nitroxide free radicals protect macular carotenoids against chemical destruction (bleaching) during lipid peroxidation.

Macular xanthophylls (MXs) lutein and zeaxanthin are dietary carotenoids that are selectively concentrated in the human eye retina, where they are thought to protect against age-related macular degeneration (AMD) by multiple mechanisms, including filtration of phototoxic blue light and quenching of singlet oxygen and triplet states of photosensitizers. These physical protective mechanisms require that MXs be in their intact structure. Here, we investigated the protection of the intact structure of zeaxanthin incorporated into model membranes subjected to oxidative modification by water- and/or membrane-soluble small nitroxide free radicals. Model membranes were formed from saturated, monounsaturated, and polyunsaturated phosphatidylcholines (PCs). Oxidative modification involved autoxidation, iron-mediated, and singlet oxygen-mediated lipid peroxidation. The extent of chemical destruction (bleaching) of zeaxanthin was evaluated from its absorption spectra and compared with the extent of lipid peroxidation evaluated using the thiobarbituric acid assay. Nitroxide free radicals with different polarity (membrane/water partition coefficients) were used. The extent of zeaxanthin bleaching increased with membrane unsaturation and correlated with the rate of PC oxidation. Protection of the intact structure of zeaxanthin by membrane-soluble nitroxides was much stronger than that by water-soluble nitroxides. The combination of zeaxanthin and lipid-soluble nitroxides exerted strong synergistic protection against singlet oxygen-induced lipid peroxidation. The synergistic effect may be explained in terms of protection of the intact zeaxanthin structure by effective scavenging of free radicals by nitroxides, therefore allowing zeaxanthin to quench the primary oxidant, singlet oxygen, effectively by the physical protective mechanism. The redox state of nitroxides was monitored using electron paramagnetic resonance spectroscopy. Both nitroxide free radicals and their reduced form, hydroxylamines, were equally effective. Obtained data were compared with the protective effects of α-tocopherol, which is the natural antioxidant and protector of MXs within the retina. The new strategies employed here to maintain the intact structure of MXs may enhance their protective potential against AMD.

The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems.

Neuromelanin is an amorphous pigment of the catecholamine origin that accumulates in certain dopaminergic neurons of the substantia nigra of human brain. In Parkinson's disease, there appears to be selective degeneration of the most heavily pigmented neurons of the substantia nigra, and this process has been linked to the presence of neuromelanin. It has been postulated that neuromelanin could increase the risk of oxidative stress reactions. On the other hand, melanin is usually considered to be an efficient antioxidant. Here we analyze experimental conditions that stimulate, or inhibit, antioxidant properties of neuromelanin. Using electron spin resonance (ESR)--spin trapping technique and salicylate hydroxylation assay, we monitored the formation of free hydroxyl radicals generated by a Fenton system in the presence of varying concentration of dopamine-melanin, a synthetic model for neuromelanin. Our data clearly indicate that the antioxidant action of neuromelanin is predominantly due to its ability to sequester redox-active metal ions such as iron. Using direct ESR spectroscopy, we have shown that ferric complexes with neuromelanin are resistant to reduction by mild biological reductants such as ascorbate. We have demonstrated that dopamine-melanin saturated with ferric ions, could enhance the formation of free hydroxyl radicals by redox activation of the ions. Thus, under the conditions that stimulate the release of accumulated metal ions, neuromelanin may actually become an efficient prooxidant. It is conceivable that neuromelanin, which normally is able to protect pigmented dopaminergic neurons against metal-ion related toxicity, could under extreme conditions have a cytotoxic role.

Photocytotoxicity of lipofuscin in human retinal pigment epithelial cells.

Lipofuscin accumulates with age in a variety of highly metabolically active cells, including the retinal pigment epithelium (RPE) of the eye, where its photoreactivity has the potential for cellular damage. The aim of this study was to assess the phototoxic potential of lipofuscin in the retina. RPE cell cultures were fed isolated lipofuscin granules and maintained in basal medium for 7 d. Control cells lacking granules were cultured in an identical manner. Cultures were either maintained in the dark or exposed to visible light (2.8 mWcm2) at 37 degrees C for up to 48 h. Cells were subsequently assessed for alterations in cell morphology, cell viability, lysosomal stability, lipid peroxidation, and protein oxidation. Exposure of lipofuscin-fed cells to short wavelength visible light (390-550 nm) caused lipid peroxidation (increased levels of malondialdehyde and 4-hydroxy-nonenal), protein oxidation (protein carbonyl formation), loss of lysosomal integrity, cytoplasmic vacuolation, and membrane blebbing culminating in cell death. This effect was wavelength-dependent because light exposure at 550 to 800 nm had no adverse effect on lipofuscin-loaded cells. These results confirm the photoxicity of lipofuscin in a cellular system and implicate it in cell dysfunction such as occurs in ageing and retinal diseases.

[Sodium chloride in food rations and dinners in mass catering institutions]. / Chlorek sodu w racjach pokarmowych i posilkach obiadowych wydawanych w wybranych zakladach zywienia zbiorowego.

The sodium chloride content in meals given by mass catering institution in all over country in 1988-1998 years was estimated. This study included daily food rations from 183 mass catering institution as hospitals, sanatoriums for both children and adults, boarding schools, infant schools and social welfare homes. We assessed also school dinners from 422 randomized selected schools and dinners from 55 internal and 56 surgical departments of provincial and regional hospitals in Poland. The mass of each meal was evaluated and sodium chloride content by Mohr's method was assessed. In most cases the salt content by 100 g of meal of 1000 kcal was calculated. The dinners and daily food rations analyze showed that sodium chloride content in meals was much higher than value recommended by World Health Organization (WHO). Salt amount in daily food rations of both children and adults was above 16 g. This value didn't include salt added to meals by boarders. School dinners provided about 7-10 g of salt. The average sodium chloride content in hospital dinners was about 16-20 g. In each studied group the NaCl content per 100 g of meal was similarly high and was 0.7-0.9 g. The results of this study show that meals given by mass catering institutions can increase risk of hypertension, strokes and gastric cancers because of high sodium chloride content.

Nitric oxide inhibition of free radical-mediated cholesterol peroxidation in liposomal membranes.

The ability of nitric oxide ((*)NO) to inhibit propagative lipid peroxidation was investigated using unilamellar liposomes (LUVs) constituted with egg phosphatidylcholine (PC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), [(14)C]cholesterol (Ch), and a nonregenerable singlet oxygen-derived primer, 5alpha-hydroperoxycholesterol (5alpha-OOH). Exposing LUVs to ascorbate and a lipophilic iron chelate at 37 degrees C resulted in an exponential decay of 5alpha-OOH and accumulation of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH), as detected by high-performance liquid chromatography with electrochemical detection. Thiobarbituric acid-reactive species (TBARS) were generated concurrently in egg PC-containing LUVs. Including the (*)NO donor spermine NONOate (SPNO, 5-50 microM) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 50-100 microM) in the reaction mixture had no effect on 5alpha-OOH decay (suggesting that iron was not redox-inhibited) but slowed TBARS and 7alphabeta-OOH accumulation in a strongly dose-dependent fashion. Decomposed SPNO or SNAP had no such effects, implying that (*)NO was the responsible agent. Accumulation of several [(14)C]Ch oxidation products, detected by high-performance thin-layer chromatography with phosphorimaging, was similarly diminished by active SPNO or SNAP. Concomitantly, a new band referred to as RCh.4 appeared, the radioactivity of which increased as a function of incubation time and (*)NO donor concentration. RCh.4 material was also generated via direct iron/ascorbate reduction of 7alpha-OOH in the presence of (*)NO, consistent with 7alpha-nitrite (7alpha-ONO) identity. However, various other lines of evidence suggest that RCh.4 is not 7alpha-ONO, but rather 5alpha-hydroxycholesterol (5alpha-OH) generated by reduction of 5alpha-ONO arising from 7alpha-ONO rearrangement. 5alpha-OH was only detected when (*)NO was present in the reaction system, thus providing indirect evidence for the existence of nitrosated Ch intermediates arising from (*)NO chain-breaking activity.

Inhibition of free radical-mediated cholesterol peroxidation by diazeniumdiolate-derived nitric oxide: effect of release rate on mechanism of action in a membrane system.

Nitric oxide ((*)NO) flux in relation to antiperoxidant action has been studied, using large unilamellar liposomes (LUVs) as target membranes. LUVs consisting of an oxidizable phosphatidylcholine (PC), [(14)C]cholesterol (Ch) as a reaction probe, and 5alpha-hydroperoxycholesterol (5alpha-OOH) as a nonregenerable primer underwent chain peroxidation when exposed to a lipophilic iron chelate [Fe(HQ)(3), 1 microM] and ascorbate (AH(-), 1 mM) at 37 degrees C. Reaction progress was monitored by (i) HPLC with reductive-mode electrochemical detection to assess the decay of 5alpha-OOH and the formation and/or turnover of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH) and (ii) HPTLC with phosphorimaging to track all major (14)C-labeled oxidation products (ChOX), including 7alphabeta-OOH, 7alpha-OH, 7beta-OH, and 5,6-epoxide. Three diazeniumdiolate (*)NO donors with different half-lives were tested for their ability to interfere with peroxidation: MANO ( approximately 1 min), PANO (15 min), and SPNO (38 min). At starting concentrations of < or =200 microM, none of the donors slowed 5alpha-OOH exponential decay, ruling out any interference with redox-active iron. However, SPNO and to a greater extent PANO (but not the decomposed donors) decreased both the initial rate and steady state of 7alphabeta-OOH accumulation in a strong dose-dependent fashion. In contrast, MANO completely inhibited 7alphabeta-OOH formation over the first 5 min of reaction, but thereafter, the peroxide accumulated rapidly, albeit more slowly than without MANO and independently of the MANO dose. The latter response diminished with increasing Fe(HQ)(3) concentration, coincident with more rapid 5alpha-OOH loss. The same general trends with MANO, PANO, and SPNO were observed when the entire population of [(14)C]ChOX species was monitored. These effects are attributed to interception of Ch- and PC-derived free radicals by (*)NO, high-flux (*)NO from MANO acting mainly on 5alpha-OOH-derived radicals (chain prevention), low-flux (*)NO from SPNO mainly on downstream radicals (chain termination), and intermediate-flux (*)NO from PANO by a combination of these mechanisms. Thus, delivery rate can be an important determinant of how (*)NO inhibits peroxide-induced lipid peroxidation.

Retinyl palmitate and the blue-light-induced phototoxicity of human ocular lipofuscin.

Lipofuscin accumulation in the retinal pigment epithelium is associated with the onset of age-related macular degeneration. Lipofuscin is phototoxic and affects cellular function through the photochemical generation of reactive oxygen intermediates. Mass spectral analysis of solvent extracts of human retinal lipofuscin granules reveals the presence of retinyl palmitate, the substrate for the enzymatic regeneration of 11-cis-retinal. Retinyl palmitate has an appreciable binding constant for phosphatidylcholine liposomes, and based on the glycophospholipids present in lipofuscin, retinal palmitate likely accumulates within the lipid content of the granule. Photochemical oxidation of retinal palmitate generates anhydroretinol, an intracellular signaling retinoid in the signal transduction cascade from the plasma membrane that causes apoptosis by generating reactive oxygen intermediates. These data are used to propose a model for the phototoxicity of lipofuscin.